The fusion oncogene bcr-abl encoded Bcr-Abl tyrosine kinase (TK) activates many pro-growth and cell survival mechanisms, which confer resistance to apoptosis. These include increased phosphorylation and transactivation by STAT-5 (signal transducer and activator of transcription), which leads to increased expression of the anti-apoptotic Bcl-xL and Pim-2 protein, as well as increased Ras/Raf/MEK/ERK1/2, AKT and NFκB activity. Through deregulated AKT activity, Bcr-Abl inhibits the Forkhead transcription regulator FOXO3a, which leads to depletion of the cyclin-dependent kinase-2 inhibitor p27 and the BH3 domain-only-containing pro-apoptotic Bim protein. Collectively, these molecular perturbations promote cell proliferation and survival and contribute to Bcr-Abl-mediated leukemia transformation of bone marrow progenitor cells. Clinical studies have shown that Bcr-Abl TK remains a therapeutic target in all phases of CML. Although highly active in inducing clinical and cytogenetic complete remissions in many CML patients, resistance to imatinib mesylate (IM, Gleevec®) is an increasing clinical problem in CML, especially in the accelerated phase (AP) and blast crisis (BC) phase where only short-term responses are observed.
The major mechanisms of resistance to IM include mutations in the kinase domain of bcr-abl, amplification of the bcr-abl gene, as well as Bcr-Abl-independent mechanisms of resistance. Within the Bcr-Abl kinase domain, close to 40 known point mutations have been described. These have been linked to IM resistance in CML. The mutations are of two broad categories: those that directly interfere with the ability of IM to bind to the kinase domain (e.g., T315I), and those that impair the ability of Bcr-Abl to achieve inactive conformation required for binding to IM, (e.g., E255K, P-loop mutation). Bcr-Abl mutations impart varying degrees of resistance to IM. Some remain susceptible to higher concentrations of IM, while others that interfere directly with the binding of Bcr-Abl to IM (e.g. T315I, involving the gatekeeper threonine residue) confer the highest form of resistance to IM. These findings highlight the need to develop and test novel anti-Bcr-Abl agents that are more potent than IM and/or are able to override the resistance to IM due to either mutations or amplifications of Bcr-Abl.
BMS-354825 (dasatinib) is a synthetic, small molecule, thiazole-based, orally-bioavailable, ATP-competitive, dual Abl/Src kinase inhibitor. Dasatinib has been shown to inhibit the activity of the Src kinase family members c-Src and Lyn. Dasatinib is able to bind the active and inactive conformations of Abl and inhibits the tyrosine kinase activity of Bcr-Abl. Dasatinib is approximately 325-fold more potent than IM in inhibiting the activity of Bcr-Abl. CrkL is a 39 kDa, tyrosine phosphorylated adaptor protein, which is involved in hematopoietic and leukemia cell signaling and is an important substrate of Bcr-Abl. Inhibition of Bcr-Abl activity in CML cells has been gauged by the decline in the levels of phosphorylated CrkL. Importantly, in vitro studies have shown that dasatinib is also able to inhibit most clinically significant IM-resistant mutant isoforms of Bcr-Abl, but is ineffective against Bcr-Abl T315I due to steric hindrance caused by the sidechain of the isoleucine. Dasatinib prolongs the survival of mice with IM-resistant, Bcr-Abl-dependent leukemia, but the drug was ineffective against tumors expressing the mutant Bcr-AblT315I. In Phase I and early Phase II studies, dasatinib has been reported to induce complete hematologic and cytogenetic responses in patients with IM-resistant or IM-intolerant chronic phase of CML. However, the responses are significantly lower in patients with more advanced phases of CML.
Vorinostat (SAHA; suberoylanilide hydroxamic acid) is a hydroxamic acid based polar histone deacetylase inhibitor. Treatment with hydroxamic acid analogue (HA) histone deacetylase inhibitors (HDIs) leads to increased levels of genes involved in cell cycle regulation such as p21 and p27, generation of reactive oxygen species (ROS), induction of TRAIL and its death receptors, as well as upregulation of the levels of the pro-death proteins, e.g., Bax, Bak and Bim. These agents are also known to deplete the levels of anti-apoptotic proteins e.g., Bcl-2, Bcl-xL, XIAP, survivin, AKT and Pim-2, in human leukemia cells. Collectively, these effects inhibit cell-cycle growth, lower the threshold to apoptotic stimuli and induce apoptosis of CML cells. Recent studies from the inventor's laboratory have demonstrated that treatment with the HA-HDIs, e.g., vorinostat, LAQ824 and LBH589, alone also depleted Bcr-Abl, as well as induced apoptosis and sensitized Bcr-Abl expressing leukemia cells to apoptosis induced by IM. By inducing acetylation of hsp90 through inhibition of HDAC6, treatment with HA-HDIs was shown to inhibit the ATP-binding and chaperone function of hsp90. This led to polyubiquitylation, proteasomal degradation and depletion of hsp90 client proteins, including Bcr-Abl, c-Raf and AKT. Significantly, the inventor's studies also showed that treatment with HA-HDIs reduced the levels of the highly IM-refractory Bcr-AblT315I and induced apoptosis of primary IM-refractory CML-BC cells.